The hawksbill turtle is an ectotherm, whose sex is determined by temperature during embryonic development. This study aimed to determine whether embryonic hawksbill turtle cells respond differently to temperature than mammalian cells. Embryonic hawksbill turtle cells were established in culture, and thermal effects on these cells were investigated in vitro. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, vitamin solution, sodium pyruvate, and 10% fetal bovine serum at 33°C and cell proliferation occurred at 25–33°C. When cells were incubated at 37°C (the temperature of mammalian cell culture) for 24 h, cell growth was completely inhibited. This growth inhibition was evidently recovered by changing the incubation temperature back to 33°C. Expression of heat shock protein was found to increase with elevating culture temperature from 25 to 33°C.